One of the factors involved in tumor-induced bone destruction is parathyroid hormone-related peptide (PTHrP). Interestingly, PTHrP is preferentially expressed in bone when compared to the primary or soft-tissue metastatic sites. While the reasons for this differential expression are unknown, it suggests that unique characteristics of the bone microenvironment contribute to PTHrP expression. Since the bone microenvironment is orders of magnitude stiffer than soft tissue, we hypothesize that the rigid microenvironment activates mechanotransduction pathways within the tumor cells. As a consequence, the Hedgehog transcription factor, Gli2, is expressed causing the expression of PTHrP and stimulating bone resorption. Our preliminary data demonstrate that tumor cells growing on rigid matrices or tissues express higher levels of PTHrP compared to cells growing on more compliant matrices. Furthermore, we have found that this process is mediated through both Rho-kinase (ROCK-1) and transforming growth factor beta (TGF-?), and that both the mechanotransduction (ROCK-1 and integrins) and TGF-? pathways are required for Gli2 and PTHrP expression. These data suggest that inhibiting mechanotransduction events will inhibit tumor cell establishment in bone at early stages. In this proposal we will investigate the mechanotransduction pathway involved in the regulation of Gli2 and PTHrP expression, and test whether inhibition of ROCK-1 and Gli2 will be effective treatments for inhibiting both early and established bone metastases. To separate the effects of rigidity from other environmental effects, we will utilize 3D matrices with tunable rigidity, pore size, and matrix protein surface concentration to measure changes in gene expression both in vitro and in vivo. Additionally, ROCK-1 and Gli2 inhibitors will be tested in the tumor-bearing mice to measure their effects on PTHrP expression and bone disease. These studies will allow us to evaluate whether mechanotransduction inhibition (early inhibition) and/or Gli2 inhibition (more established tumors) will be promising targets for the development of clinical treatment strategies for treating patient with tumor-induced bone disease.